All of the major proteins of the human erythrocyte (RBC) membrane except for protein 4.2, have been purified and characterized in great detail. Protein 4.2, which comprises approximately 5% of the total membrane protein, is known to bind to band 3, but its functional role in the membrane cytoskeleton is still largely unknown. We have recently identified an anemic patient whose RBC membranes were selectively deficient in only protein 4.2 (is less than 1% of normal amount of protein 4.2) suggesting that this protein may have an important role in stabilizing the RBC membrane. Our efforts will focus in the following areas: 1) We will determine the membrane binding site(s) for protein 4.2. Although protein 4.2 binds to band 3, recent studies by others have suggested that band 3 is not the only binding site for protein 4.2; 2) we will localize the binding domain of protein 4.2 using the techniques of selective proteolysis and membrane reassociation using purified peptides derived from protein 4.2; 3) we will examine the interaction between purified protein 4.2 and ankyrin's binding to the membrane, recent studies by us have shown that ankyrin binding to protein 4.2-deficient membranes is abnormal; 4) we will examine the structure of protein 4.2 by analyzing one and two-dimensional peptide digests of the purified protein; 5) we will determine the native state of protein 4.2 in situ and examine which other membrane proteins protein 4.2 is in close proximity to in the membrane using in situ crosslinking studies; 6) we will examine non-erythroid cells, such as platelets and brain for the presence of protein 4.2 immunoreactive analogs, preliminary studies suggest that tissue-specific isoforms of protein 4.2 may exist; 7) we will examine the synthesis of protein 4.2 in synchronized erythroid progenitor cells to determine how protein 4.2 stabilizes cytoskeletal assembly; and 8) we will examine the molecular biology of protein 4.2 with a particular emphasis on developing a cDNA probe for this protein. These studies should help to define the properties of protein 4.2 and help us to understand the functional role that protein 4.2 has in stabilizing the RBC membrane cytoskeleton. Since a deficiency of protein 4.2 is associated with RBC pathology, this work may have significant implications for understanding the pathophysiology of certain hemolytic anemias. Furthermore, the ability to probe for the genetic lesion resulting in protein 4.2 deficiency may allow us to recognize heterozygous carriers of this abnormality.